# Work with Ji
## 19/06/10
1. Prepared rice seeds by sterilizing them

a. In a 250 ml beaker I washed the seeds in a 50% bleach solution (~ 25 ml) for 20 minutes while stirring

b. Then I dumped the bleach solution and filled the beaker with water and let that stir for 15 min. (repeated 5 times)

## 19/06/11
1. I watched as Ji was planting rice seeds (1/2 will grow on a paper towel to be transferred to soil later, 1/2 will grow on turface)

For paper towel:

a. On a petri dish lined with a damp (NOT SOAKED) paper towel
b. Seeds should be placed as spread out as possible
c. Then seed are to be covered with another damp paper towel

For Turface:

d. Wash the turface by rinsing until clean (water doesn't appear cloudy) can take anywhere from 5 to 8 times
e. Wet turface was then placed it inside a small container, with a hole in the bottom that lets the water out but not the turface out
f. Cover the seeds with some extra turface
g. Spray a little extra water on top to ensure there is enough water

h. Bring both the petri dish and turface container to growth chamber 8 and place it in a bin with water, cover it

2. I washed and sterilized rice seeds, same procedure as yesterday.

## 19/06/17

1. I looked at the rice that was planted on June 10th and there were fungi growing (Ji applied a fungicide to the second batch of rice that was sterilized and it is growing)

2. I collected Arabadopsis seeds from wild type Columbia lines

a. Collect Arabadopsis from growth chamber 2
b. Clean tools and environment using 75% ethanol (including hands)
c. Collect seeds without gloves to ensure a low percentage of lost seeds
d. Fold two sheets of paper across both ways
e. Cut long and well grown arabadopsis plants from their roots (if a small arabadopsis growth is present it is unknown if the genotype is the same as the main plant)
f. Open the siliques to collect the seeds that are inside by gently twisting them open
g. Use the strainer to separate the seeds from the debris
h. Collect in an evelope or in a tube

## 19/06/18

1. To attempt to reduce fugus, the rice grown in fugicide still had fugus on it, we will atempt a new sterilization and planting technique for the rice.

a. Washed unhusked rice seeds in 75% ethanol; for 2 min by stirring the solution in a 250 ml beaker
b. Then we dumped the ethanol and washed with 50% bleach for 20 min with stirring
c. After bleach was dumped and the beaker was filled with water and rinsed for 20 minutes 5 times

2. Seed Collection of Arabadopsis Mutants:
Plants 2, 3, 4, 7, 8, 9 are SALK 087206 GLK 2 Mutants. All mutants plants labled CS9805 for GLK 1. All mutants in CS9805 plants for GLK 2
a. Same procedure as described previously for collection but this time there is cleaning of hands tables and tools between different mutants to make sure there is no contamination.

## 19/06/19

1. Learned how to work the autoclave by helping autoclave Ji's pots that he will use for maize growth

a. Generator must be above 40
b. Jacket must be around 15
c. Everything must be placed in an autoclave appropriate bin
d. If its a solid use the gravity setting (password is 1111 to select cycle)

2. I washed turface the same way as described above
3. Place the turface in the autoclave *The bin used wasnt appropriate and melted in the autoclave*
4. I repeated steps 2 and 3 with more turface and an appropriate bin
5. Prepared PCR reactions for genotyping for the first four CS829331 mutants (1-4)

Gene: CS829331
F Primer: JH54
R Primer: JH55
T Primer: Cyc28

PCR Reactions:

M1
|Reagents|Volume (µl) for one| Total volume (µl)|
|---------|------------------|-------------------|
|Water|6.55|26.2|
|Nucleotide Mix|0.4|1.6|
|JH 54|1|4|
|JH 55|1|4|
|Reaction Buffer|1|4|
|Taq Polymerase|0.05|0.2|

M2
|Reagents|Volume (µl) for one| Total volume (µl)|
|---------|------------------|-------------------|
|Water|6.55|26.2|
|Nucleotide Mix|0.4|1.6|
|JH 54|1|4|
|Cyc 28|1|4|
|Reaction Buffer|1|4|
|Taq Polymerase|0.05|0.2|

M3
|Reagents|Volume (µl) for one| Total volume (µl)|
|---------|------------------|-------------------|
|Water|6.55|26.2|
|Nucleotide Mix|0.4|1.6|
|JH 55|1|4|
|Cyc 28|1|4|
|Reaction Buffer|1|4|
|Taq Polymerase|0.05|0.2|

PCR Conditions:
|Temp|Time|
| -------- | -------- |
|94^oC| 2 min |
|94^oC|20 s|
|58^oC|40 s|
|72^oC|1 min|
|72^oC|7 min|
|12^oC|∞|
repeat steps 2 to 4 34x

6. I planted the rice seeds that were sterilized yesterday with ethanol. I planted them in the turface that was autoclaved today.

## 19/06/20

1. I ran the gel to perform the genotyping

a. To run a gel one must first mass out the agarose needed (1% of the buffer used) in this case 50 mL of 1XTAE buffer AND 0.5g agarose used.
b.Use the microwave to homogenize the solution in a wide mouth flask ~ 60 sec
c. Let cool for 2 minutes and add ethidium bromide by touching the 200µl pipette tip in the ethidium bromide and stirring it into solution
d. Once the solution is cooled to the touch (~10 min) one can pour the gel and let it harden (~20 min)
e. Mix 1µl of dye with 5µl of PCR reaction.
d. Distribute the now dyed reactions (4µl in each well) with a 100bp ladder and run the gel for 20 min

2. I did more PCR reactions:

a. Same gene as yesterday, samples: E1-H1, A2-H2, A3-E3 (17 samples)

Gene: CS829331
F Primer: JH54
R Primer: JH55
T Primer: Cyc28
Plants 5-21

M1
|Reagents|Volume (µl) for one| Total volume (µl)|
|---------|------------------|-------------------|
|Water|5.55|99.9|
|Nucleotide Mix|0.4|7.2|
|JH 54|1|18|
|JH 55|1|18|
|Reaction Buffer|1|18|
|Taq Polymerase|0.05|0.9|

M2
|Reagents|Volume (µl) for one| Total volume (µl)|
|---------|------------------|-------------------|
|Water|5.55|99.9|
|Nucleotide Mix|0.4|7.2|
|JH 54|1|18|
|Cyc 28|1|18|
|Reaction Buffer|1|18|
|Taq Polymerase|0.05|0.9|

M3
|Reagents|Volume (µl) for one| Total volume (µl)|
|---------|------------------|-------------------|
|Water|6.55|99.9|
|Nucleotide Mix|0.4|7.2|
|JH 55|1|18|
|Cyc 28|1|18|
|Reaction Buffer|1|18|
|Taq Polymerase|0.05|0.9|

b. The PCR tubes are organized as follows
|1|M1|E1|F1|G1|H1|||||
|--|--|--|--|--|--|--|--|--|--|
|2|M2|E1|F1|G1|H1|||||
|3|M3|E1|F1|G1|H1|||||
|4|M1|A2|B2|C2|D2|E2|F2|G2|H2|
|5|M2|A2|B2|C2|D2|E2|F2|G2|H2|
|6|M3|A2|B2|C2|D2|E2|F2|G2|H2|
|7|M1|A3|B3|C3|D3|E3||||
|8|M2|A3|B3|C3|D3|E3||||
|9|M3|A3|B3|C3|D3|E3||||

PCR Conditions:
|Temp|Time|
| -------- | -------- |
|94^oC| 2 min |
|94^oC|20 s|
|58^oC|40 s|
|72^oC|1 min|
|72^oC|7 min|
|12^oC|∞|

## 19/06/26

1. Helped make 3 MS solution with Ji that will be for different nitrogen tratements

a. in 2L(?) of water add:
|Reagent|Mass(g)|
|-|-|
|MS Powder|1.56|
|MES hydrate (buffer)|1|
|KCl|2.98|
|Adjust PH|5.8 g|

b. Wait 30 min and check pH again and then bottle it

## 19/06/27

1. Sample collection of shoots and roots for Ji

a. I helped by transfering samples from the cooler with liquid N₂ to the -80 freezer

b. Additionally I collected the liquid Nitrogen and brought it to the grwoth chamber this is simple, put the hose in the collection bucket and turn the nob using gloves. A lot of gas forms from the ligquid N₂ evaporation but soon you will see liquid in the container. It may be useful to fill two containers half way and then combine to get a full container because it is hard to fill one container entirely.

2. I ran the gel that was prepared on 2019-06-20. I had to wait to get restocked with 1xTAE buffer. I used the same method as described on 2019-06-20 to make the gel and run it except I used a casset with more wells.
| 150 V |
| -------- |
| 15 min|


Summary of genotypes: for CS829331
|Number|Genotype|
|--|--|
|1|Mut|
|2|Mut|
|3|Mut|
|4|Mut|
|5|Mut|
|6|Het|
|7|WT|
|8|Het|
|9|Het|
|10|Het|
|11|Mut|
|12|WT|
|13|Mut|
|14|Het|
|15|Het|
|16|Het|
|17|Het|
|18|Het|
|19|Het|
|20|Het|
|21|Het|

|Genotype|Frequency|
|--|--|
|WT|2|
|Het|12|
|Mut|7|

## 19/07/05

1. Ran PCR reactions for the genotyping of plants 1-19 of the SALK073491

Gene: SALK073491
T Primer: Cyc15
F Primer: JH42
R Primer: JH43

I used the 10mM dNTP mix

DNA used: A4-H4, A5-H5, A6-D6

M1

|Reagents|Volume (µl) for one| Total volume (µl)|
|---------|------------------|-------------------|
|Water|5.75|115|
|Nucleotide Mix|0.2|4|
|JH 42|1|20|
|JH 43|1|20|
|Reaction Buffer|1|20|
|Taq Polymerase|0.05|1|

M2
|Reagents|Volume (µl) for one| Total volume (µl)|
|---------|------------------|-------------------|
|Water|5.75|115|
|Nucleotide Mix|0.2|4|
|JH 43|1|20|
|Cyc15|1|20|
|Reaction Buffer|1|20|
|Taq Polymerase|0.05|1

M1
|Reagents|Volume (µl) for one| Total volume (µl)|
|---------|------------------|-------------------|
|Water|5.75|115|
|Nucleotide Mix|0.2|4|
|JH 42|1|20|
|Cyc 15|1|20|
|Reaction Buffer|1|20|
|Taq Polymerase|0.05|1

## 19/07/08

1. I ran the DNA from the PCR above on a gel but there where no bands.

## 19/07/10

1. Another attempt at the genotyping that would try and figure out why there were no bands

a. I diluted the DNA 4x for 4 samples (A4-H4)

b. I ran the PCR with the same conditions as above

|Temp|Time|
| -------- | -------- |
|94^oC| 2 min |
|94^oC|20 s|
|58^oC|40 s|
|72^oC|1 min|
|72^oC|7 min|
|12^oC|∞|
repeat steps 2 to 4 34x

1. 

## 19/07/23

1. I planted the seeds for CS9805 and CS9806 that will later be crossed and genotyped

a. The soil needs to be prepared 1:1 vermiculite and growth media
b.Add water but not too wet
c.Put soil in the pots (try to use all the soil to reduce waste)
d.Transfer the plants from the plates to the soil and make sure all pots are labled and watered
e.Store in growth chamber 2

## 19/08/12

1. After 2 weeks of growth Ji outlined a plan for genotyping from tissue from the CS9805 and CS9806 mutants

## 19/08/13

1. I collected tissues for the genotyping in the 96 tube blue box. It is organized accordin to the excel file in the lab summer 2019 folder on my desktop

## 19/08/14

1. I extracted DNA for the tissues extracted yesterday following the DNA extraction protocol Ji gave me

## 19/08/15

1. I used the nano drop to test DNA quality and concertration. The concertration seemed very high so I diluted it 5 fold.

3. I ran a PCR to genotype the tissues.

Primers
Posative Control: JH48 + JH49
CS9805 Amplifier: JH88 + JH89
CS9806 Amplifier: JH90 + JH91

Reactions:

|Reagents|volume (µl)|volume x 9 (µl)|
| -------- | -------- |-|
|2 x Mix|5|45|
|F Primer|1|9|
|R Primer|1|9|
|dd H₂O|2|18|
|DNA|1|
|Total|10|

Conditions using the GoTaq Enzyme:
Posative Control:
|Temp|Time|
| -------- | -------- |
|95^oC|2 min|
|95^oC|30 s|
|58^oC|30 s|
|72^oC|1 min|
|72^oC|7 min|
|12^oC|∞|
repeat steps 2 to 4 34x

This gel showed a band for every sample suggesting that the DNA extraction for samples A1-H1 were successful.

Genotyping:
|Temp|Time|
| -------- | -------- |
|94^oC|2 min|
|94^oC|30 s|
|44^oC|30 s|
|72^oC|1 min 50 s|
|72^oC|7 min|
|12^oC|∞|
repeat steps 2 to 4 34x


## 19/08/21

1. I prepared a PCR reaction to test if the DNA was viable from the rest of the DNA extraction batch using a posative control

Primers:JH48 and JH49

Reactions:

|Reagents|volume (µl)|volume x 92 (µl)|
| -------- | -------- |-|
|2 x Mix|5|460|
|F Primer|1|92|
|R Primer|1|92|
|dd H₂O|2|352|
|DNA|1|
|Total|10|

PCR Conditions:
|Temp|Time|
| -------- | -------- |
|95^oC|2 min|
|95^oC|30 s|
|58^oC|30 s|
|72^oC|1 min|
|72^oC|7 min|
|12^oC|∞|
repeat steps 2 to 4 34x

## 19/08/23

1. I ran the DNA on a gel in 2 batches:

2-7 Gel:

8-15 Gel:

Summary:
|Sample|Posative Control Band|
|--|--|
|A1|Y|
|B1|Y
|C1|Y
|D1|Y
|E1|Y
|F1|Y
|G1|Y
|H1|Y
|A2|Y
|B2|Y
|C2|Y
|D2|Y
|E2|Y
|F2|Y
|G2|Y
|H2|Y
|A3|N
|B3|Y
|C3|Y
|D3|Y
|E3|Y
|F3|Y
|G3|Y
|H3|Y
|A4|N
|B4|Y
|C4|Y
|D4|Y
|E4|N
|F4|Y
|G4|Y
|H4|N
|A5|Y
|B5|N
|C5|Y
|D5|Y
|E5|Y
|F5|Y
|G5|Y
|H5|Y
|A6|Y
|B6|Y
|C6|Y
|D6|Y
|E6|N
|F6|Y
|G6|Y
|H6|Y
|A7|Y
|B7|Y
|C7|Y
|D7|N
|E7|N
|F7|Y
|G7|Y
|H7|Y
|A8|Y
|B8|Y
|C8|Y
|D8|N
|E8|Y
|F8|Y
|G8|Y
|H8|Y
|A9|Y
|B9|Y
|C9|N
|D9|Y
|E9|Y
|F9|Y
|G9|Y
|H9|N
|A10|N
|B10|Y
|C10|Y
|D10|Y
|E10|Y
|F10|Y
|G10|Y
|H11|Y
|A11|N
|B11|N
|C11|Y
|D11|N
|E11|Y
|F11|Y
|G11|N
|H11|Y
|A12|N
|B12|N
|C12|N
|D12|N
|E12|N
|F12|N
|G12|Y
|H12|Y

Y: YES posative band
N: NO posative band

## 19/08/28

1. I changed (color coded) the excel file for the genotyping of the CS9805 and CS9806 mutants
2.